The invention relates to compositions based on derivatives of resveratrol having in particular a high stability as regards air and light.
Resveratrol (3,5,4xe2x80x2-trihydroxystilbene) exists in cis or trans form and is presented in monomer form or as the oligomer containing generally 2 to 4 monomer units.
In the following description and in the claims, the term xe2x80x9cORsxe2x80x9d will be used to designate both the monomer and the oligomers.
Study of the properties of resveratrol has allowed the demonstration of useful biological activities. Cardiovascular and anti-carcinogenic effects have thus been reported.
The practical use of ORs is hindered by difficult access, selectively, to such extracts, from plant sources which contain them. It is also due to their instability, caused by the phenol groups which they contain, and to their hydrosoluble character, which poses miscibility problems with numerous excipients generally used in therapeutics, cosmetics and in the field of foods, which on the contrary have liposoluble properties.
In order to resolve these problems, the inventors have perfected extraction methods leading to extracts which are rich in ORs, and have used protective groups of the phenol functions, which allow both a satisfactory stability to be conferred on the resveratrol monomer and oligomer and renders them liposoluble, these groups having the advantage of being eliminable in vivo.
Therefore an aim of the invention is to provide compositions based on derivatives of resveratrol monomers and/or oligomers the protective groups of which can be easily eliminated in order to release the active ingredient when desired.
It also aims to provide a process for obtaining these compositions as well as the starting monomers and/or oligomers.
Moreover the invention relates to uses of these compositions in various fields in particular in therapeutics, in cosmetics and in agro-foodstuffs.
The compositions according to the invention are characterized in that they are essentially based on esters of resveratrol monomers and/or oligomers, the monomers comprising at least one ester group of formula xe2x80x94Oxe2x80x94COxe2x80x94A, and the oligomers being formed from monomer units joined by carbon-carbon bonds, or ether, and/or monomers cross-linked by
Oxe2x80x94COxe2x80x94Rxe2x80x94COxe2x80x94 groups
A representing an alkyl radical with at least two carbon atoms, linear or branched, saturated or unsaturated, an aryl radical, except for the phenyl radical in the case of a composition of resveratrol monomer, aralkyl or aralkylene, and
R representing an alkylene radical with 0 to 10 carbon atoms, saturated or unsaturated, and/or 1 arylene radical having 1 to 3 rings and/or a heterocyclic radical, and the diastereoisomers of these units.
These compositions can be stored over a long period without alteration, in particular for at least 2 years under normal storage conditions (temperature of 10 to 22xc2x0 C., in light protective packaging, hygrometry 40-50%).
In an embodiment of the invention, the compositions are based on monomers and/or oligomers of resveratrol comprising at least one xe2x80x94Oxe2x80x94COxe2x80x94A group.
In a preferred group, A represents a saturated or unsaturated fatty acid radical.
In the case of an unsaturation, the double bonds are advantageously cis, which corresponds to the most frequent case found in the natural products. With products obtained more particularly by synthesis or hemisynthesis, the bonds are trans.
Among the fatty acids which are suitable for the implementation of the invention, the following acids can be mentioned: butyric C4:O; valeric, Cxe2x80x25:O hexanoic, C6:O: sorbic, C6:2(n-2); lauric C12:O; palmitic C16:O; stearic, C18:O; oleic, C18:1(n-9) linoleic, C18:2(n-6); linolenic, C18:3(n-6); xcex1 linolenic, C18:3(n-3); arachidonic, C20:4(n-3) eicosapentaenoic C20:5(n-3); and docosahexanoic. C22:6(n-3)
The C16 and more fatty acids are particularly appropriate as regards cosmetic uses. These fatty acids are extracted, for example, from microalgae.
In another preferred group, A represents an aryl group, except for, as specified above, the phenyl radical in the case of a composition of resveratrol monomer.
In yet another group, A represents an aralkyl or aralkylene group, the alkyl or alkylene group being more particularly C1 to C8, in particular C1 to C4. In particular the benzyl or styryl group can be mentioned.
In another embodiment of the invention, the compositions are based on monomers and/or of oligomers of resveratrol cross-linked using xe2x80x94Oxe2x80x94COxe2x80x94Rxe2x80x94COxe2x80x94 bridges.
In this structure, R represents an alkylene radical with O to 10 carbon atoms, saturated or unsaturated, and/or an arylene radical comprising 1 to 3 rings and/or a heterocyclic radical.
The cross-linked esters advantageously include as substituent R, a radical of a diacid chosen from the following acids: malic, malonic, glutaric, phthalic, a chloride of diacids, such as terephthaloyl dichloride, succinyl dichloride, sebacoyl dichloride, and adipoyl dichloride, an anhydride, or also an isocyanate such as toluene or hexamethylene diisocyanate.
In an advantageous manner, these cross-linked compositions form microcapsules or spongy masses.
The invention also relates to a process for obtaining the esters defined above.
This process is characterized in that it comprises the reaction of monomers and/or of oligomers of resveratrol with, as acylation agents, compounds of formula Axe2x80x94COxe2x80x94O-A1, or A1-Oxe2x80x94COxe2x80x94Rxe2x80x94COxe2x80x94O-A1, where
A represents an alkyl radical with at least two carbon atoms, linear or branched, saturated or unsaturated, an aryl, aralkyl or aralkylene radical,
R represents an alkylene radical with 0 to 10 carbon atoms, saturated or unsaturated, and/or 1 arylene radical having 1 to 3 rings and/or a heterocyclic radical, and
A1 represents a hydrogen atom, a halogen atom, a C1 to C8 alkyl radical, or aryl, a xe2x80x94COxe2x80x94A group or isocyanate, A and A1 not being able to represent a phenyl radical and a chlorine atom in Axe2x80x94COxe2x80x94C-A1.
The esterification reactions with acids are generally carried out at ambient temperature, in the presence of an activation agent. For example dicyclohexylcarbodiimide (DCC) or ter-butylchloroformiate can be mentioned.
The esterifications with acid derivatives are advantageously carried out according to the Schotten Baumann reaction in alkaline aqueous medium.
These reactions lead to the obtaining of esterified compositions in spongy form, which are isolated from the reaction mixture and which are purified with a view to the subsequent uses envisaged.
When diacids or their derivatives are used, an emulsion of (W/O) type is formed by dispersion, under agitation, of an alkaline aqueous solution of the monomers and/or oligomers of resveratrol in an organic solvent which is not miscible with water, then the cross-linking agent, A1-Oxe2x80x94COxe2x80x94Rxe2x80x94COxe2x80x94O-A1 in solution in said non-miscible organic solvent is added, or, as a variant, an emulsion of (O/W) type is formed by dispersion, under agitation, of an organic solution containing said cross-linking agent in an aqueous solution of monomers and/or oligomers of resveratrol, with an alkaline agent in aqueous solution added to it to adjust the pH of the dispersing phase to approximately 9-11.5.
The emulsifying agents are used at the rate of approximately 2 to 15% by weight, relative to the weight of the dispersion, in particular approximately 3 to 8%.
The appropriate agents correspond to those usually used, such as those marketed under the mark Spans(copyright) (esterified hexyl alcohols) or Tween(copyright) (esters of fatty acids and sorbitol with ethylene oxide).
Depending on the relative quantities of the aqueous and organic phases and of the emulsifying agent, an emulsion of O/W or W/O type is formed.
Agitation is carried out so as to rapidly homogenize the aqueous and organic solutions, for example by using a magnetic stirrer at 500-1000 rpm or a helix at 800-2000 rpm. The duration of this stage is generally of the order of 30 minutes.
The cross-linking occurs se product at the interface of the droplets of the emulsion.
The cross-linked esters formed are recovered, for example by centrifugation. The washed and dried products are in the form of fluid powder.
The esters formed can also be recovered by dilution of the reaction mixture using one or more solvents, decantation and/or centrifugation, and washing.
Observation by microscope shows that the droplets are in the form of approximately spherical particles, with a homogeneous size.
Their diameter can vary from approximately 25 to 300xcexc according to the conditions used for obtaining them.
In the case of microcapsules containing the active ingredients, the latter is preferably added to the aqueous or organic phase in which they are soluble.
The monomers and/or oligomers of resveratrol, abbreviated to ORs, used in the esterification stage can be obtained from various plant sources. Vitaceae, Umbelliferae, Myrtaceae, Dipterocarpaceae, Cyperaceae, Gnetaceae, Leguminosae, Gramineae, Sericeae, Haemodoraceae Musaceae, Polygonaceae, Pinaceae, Cupressaceae, Cesalpiniaceae, Poaceae, and Solanaceae can be mentioned.
In an advantageous manner, monomers and/or oligomers of resveratrol are used such as those obtained by extraction, using water and/or of an organic solvent, from vine stalks.
The process for obtaining monomers and oligomers of resveratrol, which is also envisaged by the invention, comprises the following stages
extraction by addition, to the vine stalks, of water and/or of organic solvent(s), by subjecting the whole to a treatment such as maceration/lixiviation, ultrasonics or microwaves,
delipidation before or after the extraction stage using a solvent of petroleum ether, hexane or chloroform type,
additional extraction of the extract recovered by an organic solvent of ethyl acetate or ethyl ether type,
concentration of the crude extract obtained, and, if desired, its lyophilization.
The percentage of resveratrol and of oligomers in the crude extract closely depends on the type of vine used and, for a given type of vine on the extraction method and solvents used.
According to a particularly useful aspect, considering the enrichment in ORs that it allows to be attained, the crude extract is subjected to a purification stage by chromatography. An especially satisfactory technique corresponds to centrifugal partition chromatography (CPC). This technique is in particular described by A.P. FOUCAULT, Ed., Centrifugal Partition Chromatography, Chromatographic Science Series, Marcel Dekker Inc., 1995, 68, or W.D. CONWAY, Ed., Countercurrent Chromatography apparatus theory and applications, VCH Publishers Inc., 1990.
CPC is based on the partition of the solutes between two non-miscible liquid phases prepared by the mixture of two or more solvents or solutions. One of the two phases is kept stationary by a centrifugal force.
The solvents, their proportions and the flow rate chosen closely depend both on the stability of the stationary phase within the CPC column and the actual pressure.
High-performance results have been obtained with a hexane/ethyl acetate/ethanol/water mixture with for example the respective proportions of 6/48/11/42 or 4/5/3/3.
However, it is also possible to not use hexane or to replace at least one of said solvents with an equivalent solvent on the condition of modifying its proportions.
Therefore, hexane can be replaced by saturated, even unsaturated, hydrocarbons which are apolar and non-miscible with water, such as for example heptane, cyclohexane, or also chlorinated solvents such as chloroform.
Similarly, carbonylated or carboxylated solvents can be used instead of ethyl acetate, such as acetone, methylethylketone, methylisobutylketone, methylterbutylketone.
Alcohols other than ethanol can also be used in the mixture defined above, such as for example, methanol, n-propanol, propan-2-ol, n-butanol, butan-2-ol.
The water can be replaced, at least in part, even totally, by acetonitrile.
A person skilled in the art will therefore choose the most appropriate solvent or solvents depending on the nature of the purified extract subjected to CPC.
Thanks to the invention, crude extracts and enriched fractions are therefore available containing, as majority constituents, resveratrol and/or oligomers of the latter, as shown by the chromatographs which are referred to in the examples. These different extracts, namely crude or enriched also fall within the scope of the invention.
The implementation of additional separation stages by CPC allows isolation of these extracts enriched with resveratrol monomer on the one hand, and resveratrol in oligomer form on the other hand. These separations can be carried out on fractions enriched from a crude extract or on the crude extract itself by using mixtures of appropriate solvents according to the proportions which are suitable for the sought separation.
The derivatization of the ORs according to the invention allows products of great interest in numerous fields to be made available.
The presence of the introduced ester groups confers a stability as regards air and light to the resveratrol structures. In an advantageous manner, these groups are only eliminable when they are placed under conditions where these compositions must react, which allows the properties to be exploited, in particular the anti-radical and anti-oxidant properties of resveratrol, under optimum conditions.
The innocuity of the derivatives of the invention makes them particularly useful for all applications involving an administration or a use by man or animal.
The invention therefore relates to the use of the ester compositions defined above for the production of medicaments.
These medicaments can also contain other active ingredients, in particular products with a protective effect vis-à-vis oxidation reactions. For example xcex2-carotene or vitamin E can be mentioned.
The pharmaceutical preparations produced according to the invention are in particular of use in anti-tumoral or vaso-protective treatments.
As forms of administration, the appropriate forms for the oral route are used, such as pills, tablets, gelatin capsules, or drops. These preparations advantageously contain approximately 50 to 200 mg of composition equivalent per unit dose preferably approximately 100 to 150 mg.
Other galenic forms are produced for an administration by cutaneous, sub-cutaneous, intradermic, intramuscular or intravenous route, in particular gels, solutions and others.
The compositions according to the invention can also be advantageously used for the production of cosmetic preparations.
These preparations are characterized in that they also contain at least one composition according to the invention in a proportion which allows an effective quantity of ORs to be made available, and comprising in combination the excipients allowing their application.
The liposoluble properties conferred on these preparations by the presence of the ester groups allow them to be easily incorporated with products used in a standard fashion in cosmetics.
The preparations according to the invention are presented in the form of cream, ointment, emulsion, gel, liposomes, lotion. They contain approximately 0.2 to 5% of active product.
The compositions according to the invention are also of use in the field of foods. The anti-radical properties of the ORs which they contain ensure better preservation of foods.
They can be used as additives for various products such as drinks and dairy products.
They can also be used in the form of pastes, granules or gels in various confectionery.
In these different applications, the cross-linked compositions according to the invention are moreover of use as vectors for active ingredients. These are retained in the spongy mass of the cross-linked compositions or are contained in the microcapsules.
The microcapsules can contain the active products in human or animal therapeutics, or can be used in the field of foods, in particular in dietetics.
Encapsulation allows the liposoluble or hydrosoluble character of the product to be ignored, and the various drawbacks which can be presented in the applications envisaged to be overcome. It also facilitates access to the site of action and allows the administration of the active ingredients which up to now would cause problems in this regard and/or provisionally protect them until they arrive at the site of action.
The invention will be illustrated hereafter by examples of the preparation of the esters of ORs and use for the production of medicaments and cosmetic preparations.